Real-time quantitative polymerase chain reaction (QPCR) determines, for each reaction well, the Ct, i.e. the fractional cycle number at which the well's rising fluorescence (proportional to product formation) crosses a set threshold that is several standard deviations above the baseline fluorescence (Higuchi, R., Fockler, C., Dollinger, G. and Watson, R. (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology (N Y), 11, 1026-1030). The Ct versus log(amount of input target DNA) plot is linear, allowing relative quantitation of unknowns by comparison to a standard curve derived from amplifying, in the same plate, serial dilutions of a reference DNA sample.
For many QPCR applications, the investigator wishes to normalize the signal from a target sequence (T) to the signal from a reference sequence (R). Early studies measured T and R in separate (monoplex) reactions with a dye that fluoresces upon intercalation into any double-stranded DNA, e.g. ethidium bromide or SYBR Green I, and this approach continues. More recent studies have measured T and R in the same reaction vessel, in a multicolor multiplex QPCR, using separate fluorescent dyes with distinct excitation/emission spectra for each of the DNA sequences being quantified (Wittwer, C. T., Herrmann, M. G., Gundry, C. N. and Elenitoba-Johnson, K. S. (2001) Real-time multiplex PCR assays. Methods, 25, 430-442). Measurement of T/R ratios by multiplex QPCR cuts in half the number of separate PCR reactions that must be run. Furthermore, since both T and R signals are collected within each reaction vessel, variation in the amount of a given DNA sample that is pipetted for replicate reactions no longer generates variation in the T/R ratios, as it does when T and R are measured in separate wells in monoplex QPCR.
The main disadvantage of multicolor multiplex QPCR is the relatively high cost of the fluorescent probes, and the high cost of the specialized QPCR machines that are equipped to read two or more fluorescent colors. In traditional approaches to multiplex PCR (whether or not the PCR is quantitative), it is also sometimes excessively time-consuming to identify primer sets and primer concentrations that prevent the earlier amplification of a higher copy number template by one primer pair from inhibiting the later amplification of a different, lower copy number template by a second primer pair